Locomotion changes in methamphetamine and amphetamine withdrawal: a systematic review

Despite extensive preclinical research over the years, a significant gap remains in our understanding of the specific effects of methamphetamine (METH) and amphetamine (AMPH) withdrawal. Understanding these differences could be pivotal to unveiling the unique pathophysiology underlying each stimulant. This may facilitate the development of targeted and effective treatment strategies tailored to the specific characteristics of each substance. Following PRISMA guidelines, this systematic review was conducted to examine alterations in spontaneous locomotor activity, specifically horizontal activity, in animals experiencing withdrawal from extended and repeated administration of AMPH or METH. Original articles were retrieved from four electronic databases, supplemented by a review of the references cited in the published papers. A total of thirty-one full-length articles (n = 31) were incorporated in the analysis. The results indicated that six studies documented a significant increase in horizontal activity among animals, seven studies reported decreased locomotion, and eighteen studies (8 AMPH; 10 METH) reported no significant alterations in the animals’ locomotor activity. Studies reporting heightened locomotion mainly employed mice undergoing withdrawal from METH, studies reporting diminished locomotion predominantly involved rats undergoing withdrawal from AMPH, and studies reporting no significant changes in horizontal activity employed both rats and mice (12 rats; 6 mice). Drug characteristics, routes of administration, animal models, dosage regimens, duration, and assessment timing seem to influence the observed outcomes. Despite more than 50% of papers enlisted in this review indicate no significant changes in the locomotion during the stimulant withdrawal, the unique reactions of animals to withdrawal from METH and AMPH reported by some underscore the need for a more nuanced understanding of stimulant withdrawal.


Introduction
The global prevalence of psychostimulant use, specifically methamphetamine (METH) and amphetamine (AMPH), has been on the rise (Bach et al., 2023;Castaldelli-Maia et al., 2023).Despite extensive exploration of different compounds and substances, there remains a lack of approved therapeutic agents for stimulant use disorder (Chan et al., 2019;Hazani et al., 2022).Withdrawal from prolonged stimulant use in humans is characterized by symptoms such as dysphoric mood, fatigue, sleep disturbances, increased appetite, and psychomotor changes (Zhao et al., 2021).In rodent studies, various pre-treatment regimens have been employed to replicate abstinence-related symptoms observed in humans, including anhedonia, anxiety, depression, stereotyped behavior, cognitive deficits, and psychomotor changes (Robinson and Camp, 1987;González et al., 2014;Mouton et al., 2016).
Alterations in movement serve as a sensitive indicator of the neurochemical and behavioral changes associated with drug dependence, revealing the positive and negative reinforcing effects of drugs.Drugs of abuse, through elevation of dopamine levels in the nucleus accumbens, mediate positive reinforcing effects of drugs, which initiates drug-seeking behavior.In contrary, negative reinforcement, creates a strong urge in drug-dependent individuals continue to use drugs to avoid drug withdrawal-associated adverse experience.Several locomotor changes are commonly studied in rodents within the context of drug dependence research, each providing valuable insights into the direct impact of drugs and withdrawal on the brain and behavior such as horizontal activity (distance travelled), vertical activity (rearing), and movement patterns (Iman et al., 2021).The alterations in locomotion observed during withdrawal from repeated pre-treatment with AMPH or METH can provide insights into various aspects of neurobehavior, depending on the changes observed.
The goal of this systematic review was to examine current research on alterations in spontaneous locomotor activity, with a specific emphasis on horizontal activity in animals undergoing withdrawal from extended repeated pre-treatments with AMPH or METH.

Search strategy
The systematic review included data from four online databases such as SCOPUS, Web of Science, PubMed, and Ovid MEDLINE spanning from 1946 to December 2023, with the latest search conducted on 1 December 2023.The basic search strategy involved combining keywords as follows: (amphetamine OR methamphetamine) AND (abstinence OR withdrawal) AND (locomotion).Further studies were performed by reviewing the references in the retrieved articles.

Inclusion criteria
All full-length research articles published in English that investigated changes in animals' spontaneous locomotor activity during withdrawal from repeated administrations of AMPH or METH were included.

Exclusion criteria
Case studies, case series, letters to editors, reviews, books, human studies, cell culture studies, and conference abstracts were excluded.Additionally, animal studies focusing on the acute or chronic effects of METH or AMPH without examining the withdrawal, or the impact of an acute AMPH or METH challenge on locomotor sensitization, were excluded.Studies that explored AMPH or METH withdrawal in animals without comparing locomotor differences between control animals (or baseline values) and withdrawn animals were also excluded.Studies involving animals that underwent brain surgery (e.g., microinfusion of drugs, intracranial stimulation, and brain lesions) were not considered.Furthermore, studies examining METH or AMPH withdrawal in animals that had undergone surgery or behavioral interventions (such as sleep deprivation, fear conditioning, and encounters with intruders) prior to drug pre-treatment were omitted.Studies administering other drugs (such as cocaine, heroin, alcohol, and caffeine) before METH or AMPH intake were also not included.

Study selection and article screening
Four authors (INM, RPMP, AU, and JK) independently reviewed the articles obtained from the databases.Any discrepancies were resolved through discussion to achieve a consensus.The article screening process comprised of three stages.Initially, titles were used as the basis for rejecting articles that did not meet selection criteria.Subsequently, the abstracts were reviewed to eliminate studies unrelated to AMPH or METH withdrawal and locomotion.Finally, a comprehensive examination of the full text was conducted to exclude articles that did not meet the inclusion criteria.

Results
Initially, 2,500 articles were identified across four online databases: Ovid MEDLINE (1,734), SCOPUS (461), Web of Science (148), and PubMed (157).Through title screening, 720 articles were identified (Ovid MEDLINE: 329, SCOPUS: 273, Web of Science: 62, PubMed: 56).After the removal of duplicates, 481 articles remained.This was followed by a rigorous review of the abstracts, methods, and results based on the inclusion criteria, resulting in the rejection of 452 articles.Ultimately, 28 original, full-length articles were included.Additionally, upon reviewing the references of these articles, an additional 3 articles were added, bringing the final count of selected articles to 31 (Figure 1).

Hyperlocomotion during the abstinence
Studies reporting a significant increase in the animals' locomotion during the withdrawal period mainly came from mice as experimental subjects (5 studies) and to a lesser extent, rats (1 study).In five out of six of these studies, METH was administered, and most of these studies (5 studies) used the intraperitoneal route as mode of drug administration, except for one study in which animals inhaled the drug (Rezaeian et al., 2020).
Four of these studies assessed the distance travelled to measure the animals' horizontal activity (Georgiou et al., 2016;Haidar et al., 2016;Haj-Mirzaian et al., 2018;Rezaeian et al., 2020) and the remaining two assessed total square crossing (Roohbakhsh et al., 2021) and beam crossing (Piechota et al., 2012) using behavioral apparatus such as plexiglass box, open field box, locomotion chamber, locomotor cell, wooden cage, and test cage with photocells (Table 3).

Hypolocomotion during the abstinence
In contrast to hyperlocomotion results, all studies reporting hypolocomotion during the abstinence period employed rats as their experimental subjects, males (4 studies) and females (3 studies).All these studies administered AMPH, mostly via the intraperitoneal route (5 studies), except for two studies that employed intravenous (Pulvirenti and Koob, 1993) and subcutaneous modes (Mouton et al., 2016).

No significant changes in locomotion during the abstinence
Studies that found no significant changes in the locomotion of animals withdrawn from the drug mostly employed rats (12 studies) and to a lesser extent, mice (6 studies).Eight studies administered AMPH via the intraperitoneal route, ten studies administered METH via subcutaneous (3 studies), intraperitoneal (6 studies) and water (Alavijeh et al., 2019).
Two studies investigated locomotion during abstinence from AMPH in mice.Haj-Mirzaian et al. (2018) found hyperlocomotion in NMRI mice after 5 days of 5 mg/kg AMPH administrations.However, Mandillo et al. (2003) reported no significant changes in locomotion following AMPH withdrawal.This discrepancy might be due to the lower AMPH dose used by Mandillo et al. (2.5 9).

Discussion
In our systematic review, we observed that mice, primarily those withdrawn from repeated METH administrations, displayed increased horizontal activity compared to control animals.On the other hand, rats withdrawn from repeated AMPH administrations exhibited reduced horizontal activity.This contradiction might be at least partly due to the time of day when locomotor activity was measured.As rodents are nocturnal creatures, being more active during the dark phase, the observed decrease in activity among rats could be due to their higher baseline activity during that phase.In line with this, in Forced Swim Test (FST), rats displayed less escapeoriented behavior, had lower levels of stress markers and lesser serotonin turnover in amygdala and frontal cortex when tested at night compared to day time.The results suggest that rats might be better able to cope with the stress of the test during dark phase compared to the day (Kelliher et al., 2000).Alternatively, a notable decrease in dopamine levels has been documented during the dark phase of AMPH withdrawal.This decrease might lead to reduced spontaneous movement, particularly during the night-time, without impacting spontaneous activity or dopamine turnover during daylight hours (Crippens et al., 1993).In addition, the increase in activity among mice might also be influenced by their lower baseline activity during the light phase.Furthermore, assessment duration also could have influenced the activity level.We found that studies reporting hyperlocomotion had an average assessment time of 38.3 min, while those reporting hypolocomotion had an average assessment time of 460.83 min.During longer assessment durations, animals might become accustomed to the study environment, resulting in decreased exploration or movement.However, it is also important to take note of the individual differences in animals responding to environmental stimuli.For instance, Klejbor et al. (2013) found that switching from light to dark increased activity in highly reactive (HR) rats but had no effect on low reactive (LR) rats.The finding highlights inherent differences in activity level among rodents during light/dark phase, depending on their natural tendency to explore or habituate.Moreover, studies using mice have reported an increase in locomotion in the open field (Valentinuzzi et al., 2000).Whereas, when observed in EPM, there was no significant effect of light/dark cycle manipulation on the locomotor activity of mice (Clénet, et al., 2006).The findings suggest that light influences activity level in some behavioural apparatus, such as open field, but not necessarily others.Therefore, multiple factors including the time-of-day assessment conducted, duration of assessment and type of behavioral apparatus used can contribute to the observed discrepancies in the activity level during the abstinence period.
The distinct effects of METH and AMPH withdrawal on animal locomotion is intriguing, with METH-withdrawn animals commonly displaying hyperlocomotion and AMPH-withdrawn animals exhibiting reduced locomotor activity.METH and AMPH interact with the dopamine transporter (DAT), a key target for psychostimulants.DAT plays a crucial role in clearing synaptic dopamine, affecting the strength and duration of dopaminergic signalling.Both AMPH and METH act as substrates for DAT, competitively hindering dopamine uptake (Hall et al., 2008).METH, reported to be a more potent and longer-lasting stimulant than AMPH at similar doses (National Institute on Drug Abuse, 2006), exhibits three times greater inhibition of dopamine uptake in synaptosomes compared to AMPH (Rothman et al., 2001).Moreover, in cells expressing DAT, METH more effectively triggers dopamine release than AMPH (Eshleman et al., 1994).However, contradictory findings exist (John and Jones, 2007), alongside reports indicating no discernible differences in the effects of these two stimulants (Johnson et al., 1998).In the presence of salient stimuli, METH demonstrates greater potency in increasing overall locomotor activity compared to AMPH.However, in the absence of such stimuli, their potency appears comparable (Hall et al., 2008).During abstinence, METH's absence might lead to a more pronounced decrease in extracellular dopamine levels.Consequently, hyperlocomotion may emerge as an attempt to compensate for and restore depleted dopamine signalling, a response more noticeable with METH.However, previous studies have suggested that AMPH induces greater locomotor activity compared to METH by further stimulating activity through glutamate (GLU) release in the nucleus accumbens upon acute dosing (Shoblock et al., 2003).Repeated METH administration also has been associated with a hyperglutamatergic state involving the metabotropic glutamate receptor subtype 5 (mGlu5) in the striatum (Szumlinski et al., 2017).In addition, pharmacological antagonism of mGlu5 reduces METH-induced locomotor hyperactivity (Wright et al., 2016).Currently, the differences in the effects of repeated METH and AMPH administrations on glutamatergic activity are not clear.
While studies comparing the effects of AMPH and METH in the past have primarily been conducted within the same species of rodents, this systematic review revealed differential effects between rats and mice.The vesicular monoamine transporter 2 (VMAT2), a presynaptic protein crucial for the packaging and subsequent release of dopamine and other monoamines.Notably, rat striatal vesicles exhibit a higher abundance of VMAT2 protein compared to mouse vesicles (Staal et al., 2000).This highlights potential species-specific differences in dopamine regulation.Studies employing targeted manipulation of VMAT2 gene expression levels have revealed altered animals' sensitivity to psychostimulants (Wang et al., 1997;König et al., 2020).Adult mice with reduced VMAT2 gene level expressed 25% lesser striatal dopamine content, and 40% of reduced extracellular dopamine level compared to wild type mice (Wang et al., 1997).Upon acute administration of cocaine or AMPH, mice with reduced VMAT2 expression displayed significantly higher locomotion compared to wild type mice (Wang et al., 1997;König et al., 2020).Whereas, during repeated cocaine administration, these mice showed no increase in locomotion on D8 of the treatment compared to the druginduced enhanced locomotion seen on D1 (Wang et al., 1997), suggests a complex shift in sensitivity with continued drug exposure.
In addition, studies have demonstrated that VMAT2 confers neuroprotection against METH toxicity in mice overexpressing VMAT2 (Lohr et al., 2015).This is particularly relevant in light of the observation that repeated administrations of high doses of METH lead to a decrease in VMAT2 and dopamine protein levels within the striatum (Eyerman and Yamamoto, 2007).Therefore, it is likely that the observed discrepancies in the locomotor activity may also arise from a combined effect of differential expression of VMAT2 towards the sensitivity and toxicity of METH or AMPH.
The route of administration, in addition to potency, has been shown to modulate behavioral responses to METH and AMPH.Based on the data presented in this review, pre-treatment with intraperitoneal METH generally increased locomotor activity during the abstinence period compared to the subcutaneous route.The administration of varying doses of METH, promoting locomotion (0.3 and 1 mg/kg) and stereotypy (3 mg/kg) via intraperitoneal and subcutaneous routes in Sprague Dawley (SD) rats resulted in distinctive outcomes.The highest total locomotor activity occurred notably after intraperitoneal administration at the highest dose (3 mg/kg).Conversely, the most pronounced stereotypy was observed following the highest subcutaneous dose.Furthermore, subcutaneous METH exhibited prolonged locomotor effects compared to the intraperitoneal route, despite no difference in the elimination half-life of METH between the routes.Subcutaneous administration led to a higher area under the curve for METH exposure, indicative of a slower absorption rate and sustained release.This route also displayed elevated peak concentrations of both METH and its metabolite (AMPH) compared to intraperitoneal administration (Gentry et al., 2004).Previous reports have consistently associated heightened stereotypy with higher subcutaneous METH doses, for instance profound oral stereotypy following 4.42 mg/kg subcutaneous doses of METH (Segal and Kuchenski, 1997).Stereotypy involves behaviors such as ambulation, inactive rearing, head bobbing, continuous biting or licking, circling, and continuous sniffing.As stereotypy intensifies, locomotor activity diminishes.The inverse relationship between locomotor activity and stereotypy suggests a potential explanation for reduced or no significant changes at high METH doses.The absorption of subcutaneous METH into the bloodstream occurs at a slower rate than the intraperitoneal route, resulting in a 100% bioavailability and prolonged drug effects.Conversely, intraperitoneal METH absorbs more rapidly but encounters hepatic first-pass metabolism, restricting the absorbed dose (with a bioavailability of 58%) (Gentry et al., 2004).Intraperitoneal METH administration modifies the concentration-time profile of METH and AMPH through hepatic first-pass metabolism (Sakai et al., 1983).This metabolic alteration, favoring increased AMPH formation, appears to reduce overall exposure to METH, thereby shifting the response from stereotypy to heightened locomotor effects (Gentry et al., 2004).This could possibly elucidate the heightened distance travelled by mice pre-treated intraperitoneally with METH compared to subcutaneous route.
Variations in animal strains, genotypes, gender, and age can influence the observed behavioral alterations during drug withdrawal.For example, studies indicate that C57BL/6J mice display heightened locomotor activity to AMPH (Zocchi et al., 1997;Ralph et al., 2001) and increased mesoaccumbens dopamine release compared to other mouse strains (Zocchi et al., 1997).Evaluation of striatal dopamine and metabolite levels in adult male Spontaneously Hypertensive Rats (SHR), Wistar Kyoto (WK), and SD rats showed no notable differences in baseline dopamine, homovanillic acid (HVA), and 5-HIAA (hydroxyindoleacetic acid) levels.However, WK rats exhibited lower baseline 3,4-Dihydroxyphenylacetic acid (DOPAC) levels compared to SD, hinting at potential alterations in dopamine turnover within this strain.Following an acute injection of AMPH (2 mg/kg, i. p), significant changes in DA, DOPAC, HVA, and 5-HIAA levels were observed across all strains, indicating a uniform response to the stimulant's immediate effects (Ferguson et al., 2003).AMPH administration did not impact mean adjusted delay in any of these strains (Wooters and Bardo, 2011).Additionally, compared to WK, SD rats displayed reduced 50-kHz ultrasonic vocalization following acute AMPH administration (Manduca et al., 2014), suggesting the possibility of distinct AMPH withdrawal profiles across different strains of rats.
When exposed to METH, both adult and adolescent C57BL/6J mice exhibited dopamine losses in the striatum, while adolescent DBA/2 and 129S6SvEv mice showed different responses compared to their adult counterparts (Good et al., 2011).Compared to C57BL/ 6J mice, dd mice exhibited heightened susceptibility to repeated METH administration despite both strains displaying increased ambulatory activity in response to the drug.In dd mice, reductions in both 3H-spiperone binding sites (associated with D2 receptors) and 3H-WB4101 binding sites (associated with the dopamine transporter) were observed in the striatum, cortex, and hippocampus.Conversely, C57BL/6J mice exhibited reduced binding sites only for 3H-WB4101 in these regions, indicating strain-specific variations in METH-induced neurochemical changes (Hayashi et al., 1987).These variations may link the differential effects of METH in these strains to distinct regional sensitivities to the drug.BALB/c mice demonstrated higher levels of homovanillic acid (HVA) and HVA/dopamine turnover in the striatum and frontal cortex following acute METH dosing (8 mg/kg, s. c), indicating increased dopamine release and potential neurotoxicity, suggesting heightened sensitivity to METH's adverse effects compared to C57BL/6J mice (Halladay et al., 2003).However, another study reported conflicting results, indicating that C57BL/6J mice experienced more pronounced dopamine depletion than BALB/c mice, with serotonin depletion occurring solely in male BALB/c mice compared to C57BL/6J mice.Additionally, male C57BL/6J mice exhibited greater dopamine depletion than females, while BALB/c mice did not show sexbased differences following METH treatment (Yu and Liao, 2000).These findings suggest sex-strain disparities in susceptibility to METH-induced effects.While dopamine is central to the reinforcing effects of AMPH, withdrawal symptoms are influenced by other brain regions and neurotransmitters such as serotonin and glutamate.Investigating how different strains experience changes in these and other neuromodulators could provide valuable understanding of their involvement in stimulant-induced increased sensitivity to movement during periods of abstinence.
Drug administration is stressful, especially with increased injections, as seen in studies using escalating schedules.This heightened injection stress in animals may lead to crosssensitization, potentially resulting in more pronounced locomotor reactions (Russig et al., 2005).Our systematic review's findings suggest that in studies reporting AMPH-withdrawal induced hypolocomotion, rats generally received a higher total number of injections compared to AMPH-withdrawn rats showing no significant changes in horizontal activity.Conversely, in mice, METH-withdrawn animals with increased locomotion (intraperitoneal route: 4-24) received fewer injections overall compared to METH-withdrawn animals displaying no significant changes in locomotion (intraperitoneal: 7-28, subcutaneous (1 study): 86).These observations indicate insufficient evidence to conclusively link injection stress with stimulant-induced changes in rodents' horizontal activity during abstinence.
It is crucial to acknowledge that different animal strains or genotypes may respond variably to stress, influencing the behavioral outcomes (O'Mahony et al., 2010;Marchette et al., 2018).Based on the current body of literature, it seems that rats may be more vulnerable to withdrawal stress, potentially accounting for observed differences in locomotion during AMPH withdrawal.Several studies consistently show heightened anxiety-like behavior (Vuong et al., 2010;Tu et al., 2014) and increased activity in the HPA axis (Bray et al., 2016) during AMPH withdrawal in rats.Conversely, findings related to withdrawal stress in mice are less robust.There are even reports stating that withdrawal from repeated AMPH pretreatment did not alter anxiety-like behavior in mice (Fukushiro et al., 2011).Consistent with this, several studies suggest that mice may exhibit greater resilience to stress-induced anxiety.For instance, exposure to single prolonged stress failed to induce anxiety-like behavior in mice (You et al., 2021), and adult mice do not seem to experience lasting effects following chronic stress (Barnum et al., 2012).Furthermore, mice have been noted to display reduced anxiety-like behavior in the elevated plus maze, potentially attributed to elevated neuropeptide Y levels in the amygdala, indicating a potential resistance to stress-induced anxiety (Nguyen et al., 2009).While not entirely impervious to stress, mice may showcase enhanced adaptability and flexibility in unfamiliar environments.

Conclusion
Based on the findings from studies involving mice and rats included in this review, genetic diversity and species difference can significantly impact METH and AMPH withdrawal responses.However, a majority (more than 50%) of the reviewed studies reported no significant difference in the animals' locomotion during the abstinence period.Several factors might contribute to this, such as heterogeneity in study designs and differences in withdrawal time point of assessments.Despite the insignificant results, understanding the potential for species-specific responses remains crucial as this can help researchers design their studies accordingly.Based on the reviewed studies, the findings suggest METH withdrawal primarily leads to hyperlocomotion, while withdrawal from AMPH appears to induce hypolocomotion.Translating these preclinical findings to human population is vital in understanding how prolonged METH or AMPH use leads to physical dependence during the abstinence period.Exploring the potential differences in the mechanism of action of METH and AMPH could ultimately lead to development of more targeted therapy strategies in substance use disorder.

FIGURE 1 A
FIGURE 1A summary of the literature search, screening, and selection of studies following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.

TABLE 2
Increased locomotion during the withdrawal period: Drug administration regimen.

TABLE 4
Reduced locomotion during the withdrawal period: Drug administration regimen.

TABLE 3
Increased locomotion during the withdrawal period: Behavioral assessment.

TABLE 6
No significant changes in locomotion during the withdrawal period: Drug administration regimen.

TABLE 7
No significant changes in locomotion during the withdrawal period: Behavioral assessment.

TABLE 8
Hypolocomotion versus no changes in movement: rats.

TABLE 9
Hyperlocomotion versus no changes in locomotion: mice.